Recent rapid aging of the population and increasing demand for medical services have led to a supply shortage of transfusable blood and an increase in the amount of blood preparations used. With the prevalence of diseases, such as Creutzfeldt-Jakob disease, malaria, and AIDS, there has been an increasing demand for blood that is safe against infections. Thus, the current worldwide shortage of transfusable blood causes difficulties in the surgery and treatment of patients. This blood shortage will become more serious in the future. According to data from the Korea Institute for Health and Social Affairs (2005), the shortage of blood in South Korea is estimated to be 55.5% of the required amount in 2030. The risk of transfusion-transmitted infections causes serious problems in blood recipients. Enormous costs are incurred in developing examination methods and systems to detect the infection risk but it is impossible to completely detect the infections [1]. For these reasons, there is a continued need to develop safe red blood cells that are produced in vitro.
Approximately 2.1 million units of erythrocyte preparations are used in South Korea annually. This value corresponds to at least about 4×1018 cells, as calculated under the assumption that 2×1012 cells are present in one unit. It was reported that at least 3×1019 cells are required annually to replace erythrocyte transfusion in the United States of America alone (15-million units of 2×1012 cells each). The largest bioreactor for culture of well-established animal cells, such as CHO cells, can be operated to culture cells at a maximum density of 5×107 cells in a volume of 20,000 L. 30,000 batch cultures are theoretically required at this density to produce erythrocytes in the current state of the art [2]. That is, animal cell culture methods designed for cultivation conditions in existing methods and systems, such as static culture, suspension culture, fixed bed reactors, and airlift reactors, are practically impossible to apply to the culture of erythrocytes that requires an astronomical number of cells and media and spaces large enough to accommodate the cells. Packed cell culture is the most efficient in terms of space saving. To the best of our knowledge, however, packed cell culture of erythroblasts for the production of erythrocytes has not previously been reported.
The present research team has demonstrated with mediated adhesion-related signals that erythroid cells exhibit better effects in terms of cell maturation, enucleation rate, cell viability, and myelodysplasia at a high cell density that is created by bringing mature erythroid progenitor cells into physical contact with each other to increase the production efficiency of erythrocytes [3]. Further, the present research team showed that 3D packed cell culture of erythroid cells in a tube is also effective in the production of erythrocytes. Also in other previous studies, attempts have been made to increase the density of cells per volume of the overall medium in 2D plate culture or 3D bioreactors. The present inventors have conducted the first research aimed at inducing direct contact between cells by 3D packing.
Erythroid cells in bone marrow are attached together three-dimensionally to create spaces, called erythroblastic islands, where they are mature and proliferate. Such hematopoietic spaces in bone marrow are divided in thin bony trabeculae to prevent cells from being squashed. Based on the environment of bone marrow, the present inventors have succeeded in finding an optimal packing scale for culture of erythroid cells, a pore size sufficient to support the packing scale, an optimal pore scale of a porous structure for cell culture, and a biocompatible material for the porous structure. The present inventors have also succeeded in optimizing a method for the mass production of erythrocytes by reducing the necrosis of cells caused by 3-dimensional packed cell culture and by packing cells in a spin filter for the supply of fresh media to facilitate the exchange and supply of media during cell culture. This is the first report on 3-dimensional packed cell culture of erythroid cells and is an innovative method for the production of erythrocytes, as in human bone marrow, which requires a minimum culture space and a minimum amount of media.
Papers and patent publications are referenced and cited throughout the specification, the disclosure of which is incorporated herein by reference in its entirety in order to more clearly disclose the invention and the state of the art to which the invention pertains.